This revised application will examine the role of the transmembrane domain in regulating the activity of several important receptor tyrosine kinases (RTKs), including Neu/ErbB2, FGFR3, KIT and RET. Mutations in each of these RTKs lead to significant human developmental and neoplastic syndromes. In Aim 1, model transmembrane domains will be used to study whether constitutive dimerization via the transmembrane domain regulates RTK activation. We have previously shown that a transmembrane domain consisting of the consensus repeat [VVVEVVV]n results in Neu/ErbB2 activation. Using consensus sequence transmembrane domains to provide constitutive dimerization, we will examine rotational coupling of the transmembrane domain with the kinase domain for FGFR3, KIT and RET. In Aim 2, we will explore whether rotational coupling accompanies ligand-stimulated RTK activation. Using PDGFR-beta, chosen because it can be readily activated by its ligand, PDGF-BB, we will determine whether ligand binding to the extracellular domain induces rotational translation of the intracellular domain, coupled through the alpha-helical transmembrane domain. Using a DHFR protein fragment complementation assay, as well as conventional assays for receptor activation such as transformation and autophosphorylation, these experiments will quantitate rotational coupling between the transmembrane domain and the kinase domain that may be induced by ligand. In Aim 3, we will examine the role of the juxtamembrane domain in regulating two different RTKs, RET and JUT. Mutations in the extracellular juxtamembrane domain of RET cause multiple endocrine neoplasia (MEN) and familial medullary thyroid carcinoma (FMTC), whereas mutations in the intracellular juxtamembrane domain of KIT lead to gastrointestinal stromal tumors (GIST) and mastocytoma. These experiments will thus investigate the mechanism whereby these gain-of-function mutations in the juxtamembrane domains of RET and KIT lead to receptor activation and downstream signal transduction.